本文選題：前列腺癌 + YAP��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:檢測(cè)YAP相關(guān)蛋白在不同前列腺癌細(xì)胞中具有干細(xì)胞特性的細(xì)胞的具體表達(dá)情況。利用基因干擾技術(shù),通過對(duì)YAP相關(guān)基因進(jìn)行調(diào)節(jié),觀察前列腺癌干細(xì)胞特性在體內(nèi)及離體的表達(dá)的變化。進(jìn)而探討YAP通過調(diào)節(jié)下游干細(xì)胞特性的Sox-2、OCT-4等的表達(dá),影響前列腺癌干細(xì)胞特性及自我分化作用能力。方法:實(shí)驗(yàn)包括體內(nèi)實(shí)驗(yàn)和離體實(shí)驗(yàn)。離體實(shí)驗(yàn)是利用干細(xì)胞特有標(biāo)記抗體蛋白的免疫磁珠,分選相應(yīng)具有干細(xì)胞特性前列腺癌細(xì)胞的方法,用干細(xì)胞標(biāo)記蛋白CD133抗體以及CD44抗體,分別在LNCaP細(xì)胞中篩選出具有干細(xì)胞特性前列腺癌細(xì)胞。再利用shRNA技術(shù),分別在CD133高表達(dá)CD133正常表達(dá)CD133低表達(dá)的LNCaP細(xì)胞中抑制YAP的表達(dá)。利用不表達(dá)YAP的前列腺癌干細(xì)胞進(jìn)行球形成實(shí)驗(yàn),驗(yàn)證YAP與腫瘤干細(xì)胞特性之間的密切聯(lián)系。同時(shí)檢測(cè)YAP敲除的細(xì)胞系。比較OCT4,Nanog,Cmet,CD133,CD44和Sox2的mRNA表達(dá)水平。利用人工合成的YAP突變體質(zhì)粒,即野生型YAP結(jié)合域突變體,YAP/TEAD結(jié)合域突變體,YAP/WW域突變體和SH3域突變體YAP分別轉(zhuǎn)染至C42細(xì)胞及LNCaP細(xì)胞中當(dāng)中,通過蛋白免疫印跡實(shí)驗(yàn)證明YAP與腫瘤干細(xì)胞的特有標(biāo)記物OCT4,Nanog,CD133和Sox2之間的作用機(jī)制。進(jìn)而進(jìn)一步證實(shí)YAP的結(jié)合域TEAD對(duì)YAP調(diào)節(jié)細(xì)胞的自我更新能力的作用。而體內(nèi)實(shí)驗(yàn)則是在去勢(shì)組和非去勢(shì)組TRAMP轉(zhuǎn)基因鼠體內(nèi)分別注射VP和DMSO并取瘤,研究YAP通過對(duì)OCT4,Nanog,CD133和Sox2這些腫瘤干細(xì)胞標(biāo)記蛋白的調(diào)控,進(jìn)而影響前列腺癌干細(xì)胞特性及自我更新能力。結(jié)果:1.通過檢測(cè)YAP敲除的LNCaP細(xì)胞中干細(xì)胞相關(guān)分子標(biāo)記物,可以發(fā)現(xiàn)YAP對(duì)在體內(nèi)外維持前列腺癌細(xì)胞的自我更新和去分化是必要的。2.對(duì)前列腺癌細(xì)胞系進(jìn)行插入YAP的各個(gè)結(jié)合域突變質(zhì)粒后檢測(cè)發(fā)現(xiàn),YAP調(diào)節(jié)細(xì)胞的自我更新依賴于YAP定義域的TEAD交互區(qū)域。3.在TRAMP轉(zhuǎn)基因鼠的體內(nèi)模型上,我們發(fā)現(xiàn),在阻遏YAP信號(hào)通路能夠增加ADT的治療作用。結(jié)論:YAP對(duì)在體內(nèi)外維持前列腺癌細(xì)胞的自我更新和去分化是必要的,YAP調(diào)節(jié)細(xì)胞的自我更新依賴于YAP定義域的TEAD交互區(qū)域。YAP與其下OCT4,Nanog和Sox2等腫瘤干細(xì)胞標(biāo)記物之間的相互作用,有望成為前列腺癌發(fā)展及轉(zhuǎn)移的新的關(guān)鍵分子機(jī)制。阻遏YAP信號(hào)通路能夠增加ADT的治療作用。此外,YAP有可能作為一種新的腫瘤標(biāo)記物,為臨床診治前列腺癌的新方法奠定基礎(chǔ),對(duì)判斷預(yù)后有重要作用,也提出了治療晚期轉(zhuǎn)移性前列腺癌的新的思路。
[Abstract]:Aim: to detect the specific expression of YAP associated protein in different prostate cancer cells with stem cell characteristics. The expression of prostate cancer stem cells in vivo and in vitro was observed by regulating YAP related genes by gene interference technique. Furthermore, the effect of YAP on the characteristics of prostate cancer stem cells and the ability of self-differentiation was investigated by regulating the expression of Sox-2OCT-4 and so on. Methods: the experiments included in vivo and in vitro experiments. In vitro, the method of sorting prostate cancer cells with stem cell characteristic by using the immunomagnetic beads of stem cell specific labeled antibody protein was used to label CD133 antibody and CD44 antibody. Prostate cancer cells with stem cell characteristics were isolated from LNCaP cells. Then shRNA technique was used to inhibit the expression of YAP in LNCaP cells with high expression of CD133 and normal expression of CD133 and low expression of CD133. Prostate cancer stem cells without YAP expression were used to test the relationship between the characteristics of tumor stem cells and YAP. The cell lines of YAP knockout were also detected. The mRNA expression levels of CD133, CD44 and Sox2 in OCT4 were compared. YAP / WW and SH3 mutant were transfected into C42 cells and LNCaP cells, respectively, using synthetic YAP mutants, namely wild type YAP / TEAD domain mutants, YAP / WW domain mutants and SH3 domain mutants, respectively, which were transfected into C42 cells and LNCaP cells, respectively, by transfection of YAP / WW and SH3 mutants into C42 cells and LNCaP cells, respectively. The mechanism between YAP and OCT4CT-NanogCD133 and Sox2 was proved by Western blot. It is further confirmed that the binding domain of YAP, tea, plays an important role in the self-renewal of YAP regulatory cells. In vivo, VP and DMSO were injected into the ovariectomized and non-ovariectomized TRAMP transgenic mice, respectively, and the tumor was removed. The regulation of YAP on tumor stem cell marker proteins, such as OCT4, NanogCon CD133 and Sox2, was studied. In turn, the characteristics and self-renewal ability of prostate cancer stem cells are affected. The result is 1: 1. By detecting stem cell related molecular markers in LNCaP cells knocked out by YAP, it is found that YAP is necessary to maintain self-renewal and dedifferentiation of prostate cancer cells in vivo and in vitro. After inserting YAP binding domain mutant plasmids into prostate cancer cell line, it was found that the self-renewal of YAP regulatory cells was dependent on the tea interaction region of YAP domain. In vivo model of TRAMP transgenic mice, we found that inhibiting YAP signaling pathway can increase the therapeutic effect of ADT. Conclusion it is necessary to maintain the self-renewal and dedifferentiation of prostate cancer cells in vitro and in vivo by the interaction between TEAD interaction region of YAP domain and tumor stem cell markers such as OCT4Nanog and Sox2. It is expected to be a new key molecular mechanism for the development and metastasis of prostate cancer. Blocking YAP signaling pathway can increase the therapeutic effect of ADT. In addition, YAP may be a new tumor marker, which can lay a foundation for clinical diagnosis and treatment of prostate cancer, play an important role in judging prognosis, and put forward a new idea for the treatment of advanced metastatic prostate cancer.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 于欣;喬守怡;;腫瘤干細(xì)胞研究進(jìn)展[J];中國(guó)生物工程雜志;2010年01期

，

本文編號(hào)：2092698